Composition and method for the treatment and prevention of enteric bacterial infections

ABSTRACT

The present invention provides a method of treatment or prophylaxis of enteric disease caused by Gram negative bacteria. The method includes the step of administering a vaccine or a hyperimmune material raised against said vaccine to an individual. The vaccine comprises one or more cell wall antigens reactive in a manner characteristic of O group serotypes, or reactive in a manner characteristic of lipopolysaccharide associated antigens, and at least some of said antigens are separated from bacterial cell walls or wall fragments. The invention also provides composition containing hyperimmune material as well as uses of the composition and vaccine.

This application is a national stage application under 35 U.S.C. §371from PCT Application No. PCT/AU2004/000277, filed Mar. 4, 2004, whichclaims the priority benefit of Australian Application No. 2003901008,filed Mar. 4, 2003.

FIELD OF THE INVENTION

The present invention relates to treatment and prophylaxis ofgastrointestinal disorders, to vaccines comprising antigens ofgastrointestinal pathogens and to immune materials derived from blood,milk and eggs produced when animals including cattle and poultry areinjected with the said vaccines and methods for their preparation. Inparticular the invention relates to compounds and compositions fortreatment or prophylaxis of gastrointestinal disorders, such asdiarrhoea, caused by Gram negative bacteria such as enterotoxigenic E.coli bacteria and to a method of treatment or prophylaxis of diarrhoea.

BACKGROUND OF THE INVENTION

The present invention may be used in the treatment and prophylaxis ofgastrointestinal diseases caused by a range of organisms including E.coli, Salmonella, Campylobacter, Helicobacter, Vibrio, Shigella,Yersinia and Aeromonas bacteria. For the purposes of exposition,however, the invention will be explained in its application toenterotoxigenic E. coli (ETEC) in humans. It will be understood howeverthat the broadest part of the invention is not limited by this exemplaryapplication.

Diarrhoea caused by enterotoxigenic E. coli (ETEC) causes significantdiscomfort in adults and can lead to death through dehydration of youngand older people. A significant fraction of the diarrhoea suffered bytravellers to destinations such as Mexico, Africa and South-East Asia iscaused by ETEC.

One treatment currently used for travellers' diarrhoea is prophylacticantibiotic therapy, for example with Amoxycillin. However antibioticresistance has reduced the effectiveness of antibiotic therapy andside-effects such as constipation or diarrhoea are common.

Symptomatic therapy is used for vomiting and diarrhoea, for example withloperamide hydrochloride, atropine sulphate and diphenoxylatehydrochloride. However the inappropriate use of Loperamide and Atropineleads to severe constipation and the inappropriate use of diphenoxylatemay lead to dependence. These agents are also unsuitable foradministration to children.

A further treatment includes fluid replacement therapy using isotonicdrinks. However fluid replacement therapy is merely palliative, and doesnot decrease clinical diarrhoea

Furthermore symptomatic relief and fluid therapy will treat the symptomsbut will not remove the cause.

Milk and egg products have been shown to have potential therapeutic andpreventative roles in the relief of symptoms of gastrointestinaldisorders

Peterson and Campbell in U.S. Pat. No. 3,376,198 teach the immunisationof milk-producing ungulates to produce antibodies or “protectiveprinciples” against bacteria and viruses.

Carlander et al in BioDrugs 2002; 16(6):433-7 teach the use ofantibodies derived from egg yolk to decrease Pasteurella bacteria in theopening of the gastrointestinal tract (the mouth).

Shimamoto et al in Hepatogastroenterology 2002 49(45): 709-714 teachesthe use of specific antibodies against Helicobacter pylori raised ineggs to decrease the number of H. pylori bacteria in the stomach ofpatients.

Linggood et al in U.S. Pat. No. 4,971,794 teach the use of hyperimmunebovine colostrum as a source of antibodies to E. coli. The cows werevaccinated using preparations of pili from a mixture of strains. Thepatent teaches that the vaccine must comprise antigens of a plurality ofstrains of E. coli expressing Type 1 pili, CFA 1 pili, CFA 2 pili andK88 pili. K88 is associated with porcine ETEC.

Hastings in U.S. Pat. No. 5,017,372 teaches the use of hyperimmunecolostrum from ungulates as a source of antibodies to E. coli. Theungulate vaccine was made using the following method: Various E. colibacteria were grown under conditions so that CFA 1 or CFA 2 or both wereproduced. The bacteria were then lysed by ultrasound to release theseantigens and heat labile toxins.

Freedman et al in The Journal of Infectious Diseases 1998, 177:662-7teaches the use of hyperimmune colostrum as a source of antibodiesagainst E. coli. A vaccine was made by growing bacteria strains in abroth designed to optimise CFA expression, then purifying the CFA usingprecipitation followed by size-exclusion or ion exchange chromatography.

Tackett et al in The New England Journal of Medicine May 12, 1988 pp1240-1243 describe the pooling of multiple inactivated (formaldehyde orglutaraldehyde treated) bacterial whole cell suspension in a vaccineadministered to cattle. The whole-cell suspensions comprise E. coli of Oserogroups: O6, O8, O15, O20, O25, O27, O63, O78, O114, O115, O128,O148, O153 and O159 as well as heat-labile enterotoxins, cholera toxin,CFA 1 and E. coli surface antigen 3. O antigens of E. coli such as O6,O8, O15, O20, O25, O27, O63, O78, O114, O115, O128, O148, O153 and O159are heat stable antigens located on the bacterial cell wall and not onprotruding structures such as pili (fimbriae) or flagella. These Oantigens are composed of polysaccharide moieties linked to a corelipooligosaccharide complex common to the wall material of most Gramnegative bacteria. Because of the close association between the Oantigens and the cell wall, O antigen based vaccines have been made fromcell walls or whole inactivated bacteria. The lipopolysaccharideendotoxins are a normal part of the outer cell wall of the bacteria andtheir toxic regions are embedded in the cell wall (see “Endotoxins inHealth and Disease”, 1999 Chapter 12 and other chapters). A furtherreason for the fact that normal veterinary practice uses whole cellbacterial antigens rather than individual O antigen moieties is that Oantigens are endotoxins—the use of high concentrations of endotoxins inpregnant cattle is considered problematic in prior art in terms ofanimal welfare and productivity.

The use of milk and egg products in the prior art for the prevention ofsymptoms of gastrointestinal disorders is associated with a number ofproblems.

Cholera toxin (see Tackeft et al., 1988) are likely to be difficult toregister because of their high toxicity. Further, heat labile toxins,although highly immunogenic are not likely to be protective.

The extent of protection obtained from antibodies produced as above isunsatisfactory. One of the attendant problems is that large quantitiesof immune concentrate are needed to produce a satisfactory prophylacticoutcome. When whole cell antigens are used, an enormous variety ofantibody responses arise and assays for antibody titre are difficult tointerpret. A key issue is whether a particular detected antibody isprotective or not. Whole bacterial cells have many antigens that areunlikely to be related to protection. In addition, vaccines comprisingGram negative bacteria are more likely to produce adverse vaccinereactions and therefore present regulatory problems due to adverseanimal ethics reports and therapeutic goods adverse reaction reports.Adverse vaccine reactions are likely to stop dairy farmers fromparticipating in any production venture for milk antibodies.

The discussion of the background to the invention herein is included toexplain the context of the invention. This is not to be taken as anadmission that any of the material referred to was published, known orpart of the common general knowledge in any country.

SUMMARY OF THE INVENTION

We have made the surprising discovery that enhanced treatment orprophylaxis of enteric disease caused by Gram negative bacteria inanimals and humans can be achieved by administering a vaccine or byadministering hyperimmune material raised against said vaccine, whereinthe vaccine is characterised in that it comprises one or more cell wallantigens reactive in a manner characteristic of O group serotypes orreactive in a manner characteristic of lipopolysaccharide-associatedantigens, at least some of said antigens being separated from bacterialcell walls or wall fragments.

Accordingly in one aspect the invention provides a method of treatmentor prophylaxis of enteric disease caused by Gram negative bacteria, themethod including the step of administering a vaccine or a hyperimmunematerial raised against said vaccine to an individual, wherein thevaccine comprises one or more cell wall antigens reactive in a mannercharacteristic of O group serotypes, or reactive in a mannercharacteristic of lipopolysaccharide associated antigens, at least someof said antigens being separated from bacterial cell walls or wallfragments.

In another aspect the invention provides a composition for use in thetreatment or prophylaxis of enteric disease caused by Gram negativebacteria, the composition comprising hyperimmune material prepared byimmunizing a host animal with a vaccine comprising one or more cell wallantigens reactive in a manner characteristic of O group serotypes, orreactive in a manner characteristic of lipopolysaccharide associatedantigens, at least some of said antigens being separated from bacterialcell walls or wall fragments.

In yet another aspect the invention provides a method of preparinghyperimmune material for treatment or prophylaxis of gastrointestinaldysfunction caused by enterotoxigenic Gram negative bacteria such asETEC, said hyperimmune materials being raised against a vaccinecomprising one or more cell wall antigens reactive in a mannercharacteristic of O group serotypes, or reactive in a mannercharacteristic of lipopolysaccharide associated antigens, at least someof said antigens being separated from bacterial cell walls or wallfragments.

In a further aspect the invention provides the use of cell wall antigensin the manufacture of hyperimmune material for treatment or prophylaxisof gastrointestinal disease caused by Gram negative bacteria such as E.coli (ETEC) wherein the cell wall antigens are reactive in a mannercharacteristic of O group serotypes, or reactive in a mannercharacteristic of lipopolysaccharide associated antigens, at least someof said antigens being separated from bacterial cell walls or wallfragments.

In yet another aspect the invention provides a process for thepreparation of immunoglobulins for treatment or prophylaxis ofgastrointestinal disease in animals comprising immunising a host animalwith a vaccine comprising one or more cell wall antigens reactive in amanner characteristic of O group serotypes, or reactive in a mannercharacteristic of lipopolysaccharide associated antigens, at least someof said antigens being separated from bacterial cell walls or wallfragments to thereby induce the host animal to produce hyperimmunematerial comprising the immunoglobulins.

The hyperimmune material that is produced according to the methods ofthe invention may be hyperimmune colostrum or hyperimmune colostrumextract or hyperimmune egg yolk or hyperimmune egg yolk extract.

At least some of the cell wall antigens are considered to be separatedfrom intact bacterial cell walls or cell wall fragments if, aftercentrifugation to remove whole cells and substantial cell fragments, atleast 10% of said antigens can be detected in the supernatant liquor.This centrifugation should be performed under conditions where a) saidantigens do not form micelles or other aggregated structures and b) cellwall integrity is not compromised. Preferably at least 20% of saidantigens can be detected in the supernatant liquor, more preferably atleast 30%. Preferably said antigens are substantially separated fromintact bacterial cell walls.

Another procedure that can be used to determine if cell wall antigensare separated or disassociated from intact bacterial cell walls or cellwall fragments is as follows: 1) Perform an assay on an aqueous liquorcomprising O antigens and Gram negative bacterial cell walls or cellwall fragments to determine total O antigen content per ml usingvigorous conditions which will degrade cell wall structure and release Oantigens; 2) Establish which specific concentration of Gram negativecells, grown in culture, have an O antigen content equivalent to the Oantigen content determined in step 1; 3) Measure the cell wall content(quantity) per ml of the cell culture identified in step 2. This cellwall content will naturally contain the level of O antigen measured instep 1. 4) Compare the cell wall content in step 3 with the content ofthe original liquor. If equivalent, it may be inferred that all Oantigens in the original liquor have their usual association with thecell wall. However, if the cell wall content of the original liquor isless than the cell wall content in step 3 then the original liquorcontains O antigens in excess of the expected O antigen content of cellwalls and therefore some O antigen is dissociated from the cell walls orwall fragments. If the weight of Gram negative cell wall material (perml) in Step 3 is W, then it is preferred that the weight per ml of Gramnegative wall material in the original liquor is less than 0.8 W,preferably less than 0.3 W, more preferably less than 0.1 W.

The quantity of lipopolysaccharide-associated antigen (O antigen)present in an aqueous liquor can be determined using methods known tothe art such as heating, proteinase K digestion and further heating inthe presence of phenol (see example 11). Other assays forlipopolysaccharide-associated antigens have been described by Lyngby etal. One convenient method of standardising lipopolysaccharide-associatedantigens is to compare with a cell cuture having a specified number ofGram negative organisms per ml, or a specified quantity of Gram negativewall material per ml. It may be advantageous to compare with cellcultures having 10⁴, 10⁶ or 10⁸ organisms per ml, or higher.

The quantity of Gram negative cell wall material (intact walls andfragments) present in an aqueous liquor can be determined using assaysfor specific cell wall markers known in the art. For examplepeptidoglycans are associated with the cell walls of Gram negativebacteria, and methods of analysing peptidoglycans have been described byLi et al.

DESCRIPTION OF THE FIGURES

FIG. 1 shows a silver-stained SDS-PAGE gel from the analysis of LPS froma liquor preparation. Shown on the SDS-PAGE gel are samples before andafter phenolic pretreatment. After the treatment this gel shows atypical LPS ladder. Also shown in left lane as a control is a boiledbacterial lysate.

DETAILED DESCRIPTION OF THE INVENTION

In the following detailed description of the invention we referpredominantly to the production and use of hyperimmune colostrum andhyperimmune colostrums extract that is obtained from suitably immunizedcows. However, the scope of the invention should not be taken to berestricted thereto and it is envisaged that the methods described canalso be used in the production of hyperimmune egg yolk and hyperimmuneegg yolk extract.

The preparation of hyperimmune colostrum will generally compriseimmunising a host animal, typically an ungulate animal, with a vaccinecomprising the cell wall antigens of O group serotype at least some ofwhich are separated from intact cell walls. The vaccine is used toinduce production in the host animal of immunoglobulins which arerecovered in the milk of the host.

The hyperimmune colostrum may be used in treatment or prophylaxis ofgastrointestinal dysfunction in animals including humans and non-humananimals. The colostrum is particularly useful in treatment orprophylaxis in humans including adult and infant humans. The colostrummay however be used in treatment of non-human animals including piglets.

The hyperimmune colostrum from the host animal immunized with the cellwall antigens vaccine may be administered directly in the form of milk.Alternatively the hyperimmune colostrum milk may be treated by methodsknown in the art to enrich or isolate the immunoglobulins. Thus theproduct may be whole milk, skim milk or whey protein.

The products derived from the hyperimmune colostrum may be prepared inpreparations such as food additives, aqueous solutions, oilypreparations, emulsions, gels, etc., and these preparations may beadministered orally, topically, rectally, nasally, bucally, orvaginally. The preparations may be administered in dosage formulationscontaining conventional non-toxic acceptable carriers and may alsoinclude one or more acceptable additives, including acceptable salts,polymers, solvents, buffers, excipients, bulking agents, diluents,excipients, suspending agents, lubricating agents, adjuvants, vehicles,delivery systems, emulsifiers, disintegrants, absorbents, preservatives,surfactants, colorants, flavorants or sweeteners. A preferred dosageform of the present invention is a powder for incorporation intobeverages, pills, syrup, capsules, tablets, granules, beads, chewablelozenges or food additives, using techniques known in the art.

In the case where the composition is administered as a tablet, thetablet may be made by compressing or moulding the active ingredient,with one or more accessory ingredients optionally included. Compressedtablets may be prepared by compressing, in a suitable machine, theactive ingredient in a free-flowing form such as a powder or granules,optionally mixed with a binder, lubricant, inert diluent, surfaceactive, or dispersing agent. Moulded tablets may be made in a suitablemachine, by moulding together a mixture of the powdered activeingredient and a suitable carrier, moistened with an inert liquiddiluent.

The O antigens can be separated from the bacterial cell walls byapplication of an effective amount of shear, homogenisation or heat orby effective combinations thereof. The preferred conditions used toeffect the separation can be established by carrying out the followingtest: Centrifuge the whole cell suspension which has been treated toeffect the separation and remove the whole cells and substantial cellfragments. Collect the resultant cell-free liquor and run on a gelaccording to the following protocol:

A) Analysis of LPS From Cell Free Liquors

Add an equal volume of standard phenol solution to a liquor sampleobtained as described previously, vortex and incubate in a waterbath at65° C. for 15 mins vortexing every 5 mins to denature protein in theliquor.

Centrifuge for 10 mins at 4° C. and recover aqueous phase to a freshtube.

Add 1 volume of 3× Laemmli buffer to 2 volumes of sample and separate ona 15% SDS-PAGE gel.

B) Silver Staining of LPS

The following procedure is used to preferentially stain LPS (Hitchcockand Brown, 1983):

-   (i) overnight fixation in 200 mL of 25% (vol/vol) isopropanol in 7%    (vol/vol) acetic acid-   (ii) 5 minute oxidation in 150 mL of distilled water with 1.05 g of    periodic acid and 4 mL of 25% (vol/vol) isopropanol in 7% (vol/vol)    acetic acid (solution made up just before use)-   (iii) eight 30 minute washes with 200 mL of distilled water each    time-   (iv) 10 minute silver-staining in a solution consisting of 0.1 M    NaOH (28 mL), concentrated (29.4%) ammonium hydroxide, 20% (w/vol)    silver nitrate (5 mL), and distilled water (115 mL) (make solution    just before use and stir constantly while making)-   (v) four 10 minute washes with 200 mL of distilled water each time-   (vi) 5-10 minutes developing in 250 mL of developer solution (citric    acid [50 mg], 37% formaldehyde [0.5 mL], distilled water [amount    sufficient to make 1 litre of solution] made up just before use at    an optimal temperature of 26° C. to help preferential staining of    LPS-   (vii) 1 hour in stop solution (200 mL of distilled water plus 10 mL    of 7% [vol/vol] acetic acid-   (viii) store gel in 5% glycerol and dry between cellulose sheets

In a particularly preferred embodiment of the invention the O antigensare separated from the bacterial cell walls by agitating the liquor, forinstance by using a homogeniser, typically a homogeniser comprising arotor and a stator. Preferably the ratio of the peripheral speed of therotor (in metres per second) to the rotor—stator gap (in millimetres) isin a range of 0.2 to 20, preferably 0.4 to 12, more preferably 0.8 to 6and most preferably 1 to 4. Other homogenisers may be used in which theregion of maximum shear has a shear value similar to the shear value inthe above rotor—stator gaps. In one preference the O antigens areseparated from the cell wall by heat treatment in the range 40 to 80°C., preferably 50 to 70° C. The duration of the treatment is preferably10 to 200 minutes, more preferably 30 to 60 minutes.

It is preferred that the vaccine further comprises the pilus orpilus-like antigens CFA-1 or CFA-2 or other CFAs or putative CFAs orcombinations thereof, at least some of said antigens being separatedfrom intact bacterial cell walls. These antigens may be associated withpili which have broken off from the cell wall. Methods of preparing saidseparated antigens or pili have been described in the art, for exampleLinggood in U.S. Pat. No. 4,971,794.

It is preferred the bacteria from which each type of O antigen isisolated are grown in separate bacterial culture systems, and afterseparation of the O antigen from the bacteria, the component antigensare added together to form a component of the vaccine.

It is preferred that the serotype O antigens in the vaccine are chosenfrom the set: O6, O8, O15, O25, O27, O63, O78, O114, O115, O128, O148,O153, O159, and other serotype O antigens associated withenterotoxigenic E. coli. Most preferably the serotype O antigens in thevaccine comprise O78.

The liquor containing the separated O antigen may contain extraneousmaterial. In one preference the liquor is further purified by ammoniumsulphate precipitation of extraneous proteins, which involves graduallyincreasing the ammonium sulphate concentration from 0 to 20%, andcentrifuging the extraneous material.

In one preference the separated O antigen is further concentrated. Inone preference to the above liquor at 20% ammonium sulphate, furtherammonium sulphate is added to a final concentration of 40% andcentrifuged to provide O antigens in the precipitate. The ammoniumsulphate can be removed by resuspension and dialysis, for examplethrough a membrane having a cut-off size in the range 1,500-10,000molecular weight units (Daltons).

The dialysed material is preferably constituted in the range of 0.1 to10 mg/ml of protein preferably 0.5 to 3 mg/ml of protein, and added to avaccine adjuvant such as MONTANIDE (sold by Seppic, Paris, France),Aluminium Hydroxide (available from Sigma Chemical Company, St Louis,Mo., USA) or QuilA (an ISCOM consisting of antigen modifiedsaponin/cholesterol micelles and described in U.S. Pat. No. 6,383,498)or other vaccine adjuvants known to the art.

In one preferred form of the invention the adjuvant used to prepare avaccine emulsion is “MONTANIDE ISA 206”, an NRA approved veterinaryadjuvant made by Seppic and supplied by Tall Bennett in Australia.MONTANIDE ISA 206 is an immunostimulant composition containing esters ofoctadecenoic acid and anhydromannitol in an oily solution. It has beendeveloped for the manufacture of mixed oil vaccines and istoxicologically controlled and of high purity. This adjuvant allows thepreparation of a mixed type “water in oil in water” (w/o/w)immunogen/adjuvant emulsion, which is effective, well tolerated, veryfluid and easy to inject. This type of formulation produces a rapidimmune response from the immediately available antigens present in theaqueous phase and a durable immune response from the antigen confined inthe oily phase.

In one preference one volume of constituted dialysed material is addedto 0.3 to 3 volumes of vaccine adjuvant, preferably 0.7 to 1.5 volumesof vaccine adjuvant. Many methods of analysing for protein are known tothe art, for example the Lowrey protein assay (J. Biol Chem 1951, 193:265-275).

The vaccination regimen leading to the production of hyperimmunecolostrum preferably involves the injection of an animal with 0.3 to 15mL of vaccine on 2 to 8 occasions prior to parturition. The time periodbetween successive vaccinations is 1 to 4 weeks, more preferably 2 to 3weeks. Colostrum is derived from mammals, preferably ungulates, morepreferably dairy cows. Methods for production and processing ofcolostrum are provided in U.S. Pat. No. 5,780,028 the contents of whichare incorporated by reference.

The processed hyperimmune colostrum can be formulated as a tablet or asa powder within a capsule or as an additive to a drink mix as describedin U.S. Pat. No. 5,780,028.

Preferably the formulation comprising hyperimmune colostrum furthercomprises a food-grade antimicrobial moiety, such as citrus extracts andiodine based antiseptics. In one preference the antimicrobial moiety isthe grapefruit seed extract of the chemical family diphenolhydroxybenzene sold under the product name CITRICIDAL by NutriBiotics ofRipton, Vt., USA.

A preferred hyperimmune colostrum for an oral dose formulation comprisesantibodies against a plurality of antigens. Said preferred hyperimmunecolostrum is made by separating said antigens into two or more vaccinelots and vaccinating two or more herds of cattle with individual lots,and blending the colostrum from the herds.

In one preference the vaccine will comprise E. coli O antigens chosenfrom the set consisting of O6, O8, O9, O15, O20, O25, O27, O29, O03,O63, O64, O78, O88, O105, O114, O115, O126, O127, O128, O148, O153,O159.

Preferably one vaccine lot will comprise E. coli O78 antigen and CFA1pilus antigen, and the other vaccine lot will comprise antigens chosenfrom the set consisting of O6, O8, O15, O25, O27, O63, O78, O114, O115,O128, O148, O153, O159 serotype O antigens and CFA 2 pilus antigen.

In one preference the serotype O antigens are chosen from the set O6,O8, O78, O128.

While in its broadest aspect the invention may be applied in thistreatment or prophylaxis of gastrointestinal disease caused by a rangeof organisms it is particularly suited to treatment of gastrointestinalinfections of enterotoxigenic E. coli and particularly in treatment orprophylaxis of traveller's diarrhoea. Accordingly in a particularlypreferred embodiment the invention relates to a method or use as definedabove wherein the Gram negative bacteria is E. coli. In the mostpreferred embodiment the method is used in prophylaxis or treatment ofdiarrhoea.

The invention will now be described with reference to the followingexamples. It is to be understood that the examples are provided by wayof illustration of embodiments of the invention and that they are in noway limiting to the scope of the invention.

Example 1 Manufacture of a Single Strain E. coli Vaccine

This example describes the preparation of a vaccine according to theinvention in which the wall antigen is O78 and the pilus antigen isCFA1. The procedure is as follows.

Day 0

(Step A) Strain Rejuvenation

The strain to be rejuvenated is E. coli H10407 (Taurchek et al, PNAS USA2003, 99:7066-7071). Take 2 CFA plates (Evans et al, Infect Immun 1977;18:330-337) from the media refrigerator and place them in the biologicalsafety cabinet. Remove the vial containing E. coli H10407 from theliquid nitrogen tank and place it in the biological safety cabinet. Openthe vial and use a sterile loop to remove a small quantity of frozenmaterial. Streak this material onto CFA plates. Place the “rejuvenationplates” in the 37° C. incubator overnight under aerobic conditions.

Day 1

(Step B) Inoculation of “Starter Suspension”

Examine each “rejuvenation plate” for pure growth. If pure growth ispresent proceed.

Working in the biological safety cabinet, remove several colonies fromone “rejuvenation plate” with a sterile loop and inoculate a McCartneybottle containing 20 mL of phosphate buffered saline (PBS) pH 7.2. UseMcCartney bottles and PBS that have been sterilised by autoclaving.

(Step C) Inoculation of “Vaccine Plates”

Inoculate 50 μL of “starter suspension” onto each of multiple CFA plates(microbiological nutrient plates formulated to enable production ofCFA).

CFA plates are prepared using 1% casamino acids (BD Difco) and 0.15%yeast extract (Oxoid) in 2% agar containing 0.005% MgSO₄ (anhydrous) and0.0005% MnCl₂ (tetrahydrate), as described in media preparation (Evanset al, Infect Immun 1977; 18:330-337). Place the “vaccine plates” in the37° C. incubator for 18-24 hours under aerobic conditions.

Day 2

(Step D) Haemagglutination Test on “Vaccine Plates” to Test for PilusProduction

Carry out the Haemagglutination test (Evans et al, Infect Immun 1977;18:330-337). Test one “vaccine plate” for each strain to be used in thebatch. If positive proceed.

(Step E) Washing of “Vaccine Plates”

Remove the “vaccine plates” from the incubator. Check each one forcontamination and reject any affected plates.

Working in the biological safety cabinet, use 1.5-2.0 mL of sterile 0.1Msodium phosphate buffer (pH 7.2) to wash the bacterial growth from thesurfaces of the “vaccine plates” into a sterile Schott bottle. Pre-coolthe buffer on ice before use. Add sodium azide to a final concentrationof 0.05% to the “vaccine washings”.

Keep the “vaccine washings” on ice for at least 30 minutes beforecommencing homogenisation.

(Step F) Enumeration of “Vaccine Washings”

Carry out enumeration of the “vaccine washings”. Ensure that thematerial has been thoroughly agitated to disperse all clumps and thatthe dilutions chosen are appropriate to the degree of concentration ofbacterial cells in the washings for the batch being manufactured.

(Step G) Purity Sampling

Assemble sufficient materials for purity testing each “vaccine washing”(i.e.; 3 HBA Plates, 3 TSA Plates and 3 MAC plates).

Working in the biological safety cabinet, streak out each “vaccinewashing” onto 3 HBA, 3 TSA and 3 MAC plates, plating for singlecolonies. Place the plates in the 37° C. incubator under aerobicconditions.

Day 3

(Step H) First Reading of the “Purity Test Plates”

Carry out the first reading on the “purity test plates”. If the platescontain only colonies of E. coli proceed.

(Step I) Homogenisation of “Vaccine Washing”

Homogenise the “vaccine washing” in the homogeniser for a total of 15minutes at one minute intervals, with one minute of cooling in anice-bath between each interval. Centrifuge the “homogenised vaccinewashing” in the high speed centrifuge at 12,000×g for 20 minutes at 4°C. Keep the supernatants (HVW super 1) and store at 4° C. for 1-3 days.

Day4

(Step J) Second Reading of the “Purity Test Plates”

Carry out the second check on the purity test plates. If the platescontain only pure colonies of E. coli proceed.

Day 6

(Step K) Separation of LPS Fraction

Centrifuge the “HVW super 1” in the high speed centrifuge at 12,000×gfor 20 minutes at 4° C. Keep the supernatant (HVW super 2).

Add sterile saturated ammonium sulphate to the “HVW super 2” slowly over1 hour until 20% saturation is reached. Stir the “HVW super 2” on amagnetic stirrer while adding the saturated ammonium sulphate. At theend of the hour allow the material to equilibrate for 30 minutes.

Centrifuge the “HVW super 2” in the high speed centrifuge at 12,000×gfor 20 minutes. Keep the supernatant (HVW super 3).

Add sterile saturated ammonium sulphate to the “HVW super 3” slowly over1 hour until 40% saturation is reached. Stir the “HVW super 3” on amagnetic stirrer while adding the saturated ammonium sulphate. At theend of the hour allow the material to equilibrate for 30 minutes.

Centrifuge the “HVW super 3” in the high speed centrifuge at 12,000×gfor 20 minutes. Keep the pellet (LPS fraction). Resuspend the “LPSfraction” in cold 0.05M sodium phosphate buffer pH 7.2 at a ratio of 10mL buffer for each 250 CFA plates that were used to produce the “vaccinewashing” from which the “LPS fraction” was originally derived.

(Step L) Dialysis of “LPS Fractions”

Dialyse the “LPS” fraction, using a 3,500 MW cut-off membrane, for 24-48hours at 4° C. against 250-1,000 volumes of cold 0.05M sodium phosphatebuffer pH 7.2. Change the buffer every 2-8 hours during dialysis. Whencomplete, keep the “LPS dialysate” on ice until ready to assay theprotein content.

Day 7

(Step M) Assaying Protein Content of “LPS Dialysate”

Use the Lowry protein assay to measure the protein content of each “LPSdialysate”. On the basis of the results dilute each “LPS dialysate” sothat it contains 1 mg/mL of protein in 0.05M sodium phosphate buffer pH7.2, and store in a sterile Schott bottle.

(Step N) Inactivation of Vaccine

Add formaldehyde to each “LPS dialysate” so that the final concentrationof formalin is 0.3%. Store the “formalinised LPS dialysate” at 4° C. for3 days.

Day 10

(Step O) Sterility Checking Part 1

Carry out a basic sterility check on each “formalinised LPS dialysate”by inoculating 0.5 mL of each into 3 TSB tube broths. Place the“sterility check tubes” in the 37° C. incubator under aerobicconditions.

Day 14

(Step P) Sterility Checking Part 2

Check the “sterility check tubes” for absence of growth. If pure growthis absent proceed.

(Step Q) Storage of “Formalinised Pilus/LPS Dialysate”

For longer term storage of the “formalinised LPS dialysate”, place it atminus 20° C.

Day 14 or Later

(Step R) Adjuvanting Stage One

Bring the “formalinised LPS dialysate” to 30° C. by placing it in awater bath. At the same time bring an equivalent volume of the adjuvant(MONTANIDE ISA 206) to 30° C. in a water bath.

Pour the adjuvant into a large beaker which has been sterilised byautoclaving (S13). Use the Ika laboratory mixer with the 3-blade paddleto stir the adjuvant at 200 RPM. Add the “formalinised LPS dialysate”gradually over 2 minutes. Increase the speed to 2,000 RPM and maintainfor 10 minutes.

Store at 4° C. for 24 hours.

Day 15 or Later

(Step S) Adjuvanting Stage Two

The day after Step L above, bring the “1^(st) stage adjuvanted vaccine”to 30° C. in a water bath. Pour the warmed material into a large beakerwhich has been sterilised by autoclaving (S13). Use the Ika laboratorymixer with the 3-blade paddle to stir the material at 200 RPM for 2minutes. Increase the speed to 2,000 RPM and maintain for 10 minutes.Store at 4° C. for 24 hours.

(Step T) Checking Quality of Emulsion

The day after Step M above, use a sterile Pasteur pipette to take asmall aliquot of the “2nd stage adjuvanted vaccine”. Fill a 250 mLbeaker with approximately 200 mL of water. Place a drop of the aliquotonto the surface of the water. If the drop partially dilutes itself,giving a milky appearance to the water then it is water-in-oil-in-waterand is acceptable. Store at 4° C. for 24 hours.

Day 16 or Later

(Step U) Filling

Working in the biological safety cabinet, use a funnel and measuringcontainer which have been sterilised by autoclaving to measure out theappropriate volume of the vaccine. For 250 mL pillow packs this is253-255 mL Pour this material into pillow packs which have beensterilised by gamma radiation. Rubber stoppers and metal caps that havebeen sterilised by autoclaving are then used to close the top of eachpillow pack. This process is then repeated until the appropriate numberof pillow packs for the batch have been filled.

(Step V) Retention Sampling

Procure a Retention Sample.

(Step W) QC Sampling for Sterility Testing and Free Formalin LevelTesting

Using the material left over at the end of the filling run, fill a 20 mlsample into a MacCartney bottle which has been sterilised byautoclaving. Use the same funnel and measuring container which were usedto fill the rest of the run. Use this sample to perform sterilitytesting and free formalin level testing.

(Step X) Labelling

Label each batch.

Store in refrigerator.

(Step Y) First Sterility Check

Four days after filling, carry out the first check on the “sterilitytest tubes” and “sterility test plates” as described in Testing forSterility.

(Step Z) Second Sterility Check

Seven days after filling, carry out the second check on the “sterilitytest tubes” and “sterility test plates” as described in Testing forSterility.

(Step AA) Third Sterility Check

Eleven days after filling, carry out the third check on the “sterilitytest tubes” as described in Testing for Sterility.

(Step AB) Fourth Sterility Check

Fourteen days after filling, carry out the fourth check on the“sterility test tubes” as described in Testing for Sterility.

(Step AC) Product Use

The batch is acceptable for use if it satisfies the followingspecifications:

-   -   Physical appearance: a milky creamy liquid in a plastic pillow        pack labelled with the approved format label including the name        “Anadis E. coli Vaccine”.    -   Purity: presence of a pure growth of E. coli on all purity test        plates for each strain at the time of both first and second        purity checks.    -   Sterility: absence of any indication of growth on plates or in        tubes at the time of the sterility check and initial formal        sterility test, or absence of any sign of growth during        retesting as described in Testing For Sterility.    -   Potency: Greater than or equal to 1.0 mg of protein per mL of        finished product.    -   Free Formalin Level: level in the QC sample is no greater than        0.002% W/V.    -   Emulsion quality: a drop of the emulsified vaccine placed on        water partially dilutes itself, giving to the water a milky        appearance.

Example 2 Preparation of Hyperimmune Colostrum

The vaccine prepared according to Example 1 was used for the preparationof hyperimmune colostrum as follows.

Cows are immunised, by a registered veterinarian, with a 2 ml injectionof an emulsion of the vaccine in adjuvant into the muscle tissue on theside of the neck. Up to 5 injections are given at 2 weekly intervalsduring months 6 to 8.5 of gestation, ceasing 1 month before parturition.Test bleeds are taken from a selection of the immunised cows and assayedto determine the level of specific antibodies. Results of these assaysare used determine if a satisfactory titre has been achieved.

To limit the possibility of self-inoculation, immunisations are onlymade by a registered veterinarian, with assistance from experiencedcattle handling personnel. During immunisation, cows are suitablyrestrained e.g. in a race, a crush or in a rotary milking carousel, togive clear access to the neck muscle. The injection site is clipped anddisinfected before immunisation and subsequent injections are given onalternate sites in the same animal.

The method of manufacture after collection of the raw hyperimmunecolostrum from the first milking is described below.

Example 3 Vaccine Preparation

This example describes the preparation of a vaccine according to aprocedure typical of teachings of the prior art.

Bovine vaccines and the colostral antibodies against E. coli strainH10407, pilus antigens, and toxins were prepared according to themethods described below.

Preparation of Colostral Antibodies.

The antibodies were isolated from the colostrum of dairy cows immunizedwith enterotoxigenic E. coli strain H10407 and a selection of pilusantigens and heat-labile enterotoxin (LT). The cows under veterinarysupervision and were housed in an experimental dairy farm. See J. Husuet al (1993).

Vaccine Preparation.

Bacterial antigens for the production of the vaccines were prepared fromthe enterotoxigenic E. coi strain H10407 (078:H11 CFA/I⁺ ST⁺ LT⁺). Threeantigens were purified from this strain:

-   -   (i) formalin-inactivated suspension of whole cells (10⁹ bacteria        per ml);    -   (ii) heat-labile enterotoxin (2 mg/ml)    -   (iii) CFA/I (2 mg/ml)

Vaccines were made by emulsifying an equal volume of each antigen withthe same volume of Freund's incomplete adjuvant.

In addition to the above antigens, a commercial oil adjuvant vaccinecontaining purified pilus antigens from E. coli K88ac, K99 and 987P (E.coli Vaccine; Commonwealth Serum Laboratories Ltd.) was used.

Processing of Colostrums and Isolation of Whey Immunoglobulins.

Colostrum milk was collected from immunized cows during the first weeksof lactation. The milk was collected into individual containers and keptfrozen until processing. Briefly, the milk fat was removed bycentrifugation and skim milk was pasteurized at 56° C. for 30 minutesand then coagulated by renetting. After removal of milk curd containingcasein, whey was centrifuged and the fine precipitate discarded. Anequal volume of saturated ammonium sulphate solution was slowly added tothe whey with continuous missing. After centrifugation, the resultingprecipitate was saved and the supernatant containing lactose and saltswas discarded.

The precipitate was dissolved in 0.32 M NaCl in 30% of original volume.This solution was extensively dialyzed against 5 volumes of saline usingAmicon apparatus with the spiral membrane SIY 30 cartridge.

The antibody solution was concentrated to 10% solids, snap frozen, andfreeze-dried. Finally, the freeze-dried antibody powder was sterilizedby γ irradiation (25 KGrays). See Hilpert (1984), Antibodies—Alaboratory Manual (1988) and Cravioto et al (1982).

Example 4 Determination of Cell Wall Separated O Antigens

This example provides a method for generating 10⁶ cfu/ml Gram negativebacteria, for removing O antigens from these bacteria and fordetermining the quantity of O antigens derived from the cell wall ofthese bacteria. The methods of this example enable the calculation of an“LPS-bacterial equivalent” —this is the amount of bacteria that willcontain a given quantity of LPS. In the example below, 3 mg per ml ofLPS is equivalent to 10⁶ cfu/ml of ETEC Gram negative bacteria.

Reagents

Phosphate Buffered Saline (PBS)

SDS-PAGE lysis buffer (20 ml) prepared as follows:

[FINAL] 2M Tris-HCl pH 6.8 10.0 ml  (1M) dH₂O 2.4 ml 10% SDS 4.0 ml (2%)glycerol 2.0 ml (10%) 0.05% bromophenol blue 0.8 ml (0.002%)

-   -   Add 40 μl 2-mercaptoethanol per 960 μl buffer just before use        (4%)        Procedure

-   1. Bacterial strains are grown overnight at 37° C. on Nutrient Agar    (NA) plates.

-   2. The following day bacteria are harvested into PBS and made up to    a 0.5 McFarlane equivalent (1×10⁸ CFU/ml).

-   3. The bacterial suspension is then diluted 100 fold in PBS to give    1×10⁶ CFU/ml.

-   4. This preparation is dispensed into 1 ml aliquots in sterile    eppendorf tubes and centrifuged at 13 000 rpm for three minutes to    pellet the bacteria.

-   5. After centrifugation the supernatant is discarded and the pellet    is resuspended in 50 μl of SDS-PAGE lysis buffer.

-   6. The samples are then boiled for ten minutes, cooled on ice    briefly and pulsed in the centrifuge to collect condensation.

-   7. 2.5 μl of proteinase K (20 mg/ml) is added to each tube and    incubated in a water bath at 60° C. for one hour.

-   8. After incubation an equal volume of phenol is added to each tube,    vortexed and further incubated in a water bath at 65° C. for 15    minutes, vortexing every five minutes.

-   9. The sample is then centrifuged for ten minutes at 4° C. and the    aqueous phase is recovered to a fresh tube for quantification of    LPS.    Quantification of LPS

The Chromogenic Limulus Amebocyte Lysate (LAL) test is a quantitativetest for Gram negative bacterial endotoxin. A sample is mixed with theLAL supplied in the kit and incubated at 37° C. for ten minutes. Asubstrate solution is then mixed with the LAL sample and incubated at37° C. for a further six minutes. The reaction is stopped with 10% SDS.If endotoxin is present in the sample a yellow colour will developmeasured at an absorbance of 405 nm. The amount of endotoxin in thesample is measured with the use of an endotoxin standard curve.

-   1. Phenol extracted LPS sample is diluted to 1 ml in LAL reagent    water, and further dilutions prepared for assay in the LAL assay (    1/10, 1/50, 1/250, 1/1250)-   2. Endotoxin standard dilutions are prepared for standard curve (3    μg/ml-0.02 μg/ml) by making two fold serial dilutions in LAL reagent    water.-   3. Method for 96 well tray:

Sample Blank Test sample or endotoxin standard 50 μl — LAL reagent water— 50 μl LAL 50 μl 50 μl Mix and incubate at 37° C. for ten minutes.Chomogenic substrate 100 μl  100 μl  Mix and incubate at 37° C. for afurther six minutes. Stop buffer (10% SDS) 50 μl 50 μl

Read OD 405 nm on ELISA plate reader.

-   4. The OD measurements of the standard curve are then graphed (OD vs    endotoxin concentration).-   5. The unknown sample concentrations are then calculated by reading    the OD off the standard curve and calculating the amount of    endotoxin present.

Example 5 Treatment of Humans using Hyperimmune Colostrum Extract

Protection from diarrhoea caused by enterotoxigenic Escherichia coli(ETEC H01047) by oral treatment with a tablet preparation of bovinecolostrum extract sourced from cattle vaccinated against bacterialantigens and toxins.

Tablets were manufactured by Anadis Limited from the colostrum of dairycattle immunised against multiple bacterial antigens and toxins. Twodouble blind control trials involved a total of 90 healthy volunteersand tested the protection given by forms of the tablet (and placebo)against a challenge dose of 1-2×10⁹ E. coli O78.

The first study, with 30 participants, evaluated the effect of tabletscontaining 400 mg of colostrum extract taken with sodium bicarbonatebuffer. There was 93.4% protection in the treatment group when comparedto the placebo group of 26.7%. The second study tested tabletscontaining 400 mg colostrum extract given with buffer (85.7% protection)or without buffer (80% protection) and 200 mg colostrum extract givenwithout the buffering drink (64.3% protection)—compared to the placebogroup of 14.3%.

The active tablet formulations were significantly (p<0.001) better thanthe placebo treatments at protecting the volunteers from clinical signsof diarrhoea after challenge with pathogenic doses of enterotoxigenic E.coli. The studies suggest this tablet formulation of bovine colostrumextract containing antibodies against multiple enterotoxigenic bacteriaand toxins has the potential to prevent clinical diarrhoea associatedwith these bacteria. The difference between buffered and unbufferedtreatments was not statistically significant.

Example 6 Treatment of Humans using Hyperimmune Colostrum

This example describes protection in human trial using a colostrumtablet made from hyperimmune colostrum induced by the vaccine describedin Example 3.

Twenty two healthy adult volunteers were recruited and enrolled for adouble blind challenge trial. Half were given E. coli specific bovineimmunoglobulin tablets with bicarbonate solution or Skim milk placebotablets with bicarbonate solution. The volunteers took their allottedtreatment three times a day for 7 days. On the seventh day a challengeof 1×10⁹ E. coli strain H010407 (ETEC O78) was given as a drink to allparticipants. The participants were then observed for signs of ETECinduced gastrointestinal disease.

Attack rate for diarrhoea in the groups was: 4/11 in the product group,7/11 in the placebo group (p=0.2). The treatment (hyperimmune colostrumtablet) provided 44% protective efficacy against diarrhoea caused byETEC H010407 when compared to the placebo group.

Example 7 Procedure of Antibody Titre Determination

This example describes an Elisa assay in which whole E. coli cells arecoated on the microtitre plate. The assay is an antibody titre assay,and colour development is catalysed by goat anti-bovine IgG—peroxidaseconjugate.

0.5 micrograms of heat-killed E. coli cells in 100 μlcarbonate-bicarbonate coating buffer was dispensed into each well of a96-well Maxisorp Immuno-plate (Nunc, Roskilde, Denmark), and left at 4°C. overnight. Plates were washed 6 times in PBS-0.05% TWEEN(polyoxyethylene (20) sorbitan monolaurate (C₅₈H₁₁₄O₂₆)) buffer,comprising 137 mM NaCl, 1.5 mM KH₂PO₄, 8 mM Na₂HPO₄, pH 7.4. 100 μl ofeach test serum or colostrum, diluted in PBS-TWEEN (polyoxyethylene (20)sorbitan monolaurate (C₅₈H₁₁₄O₂₆)) containing 12 mg/ml casein, wereadded to each well and incubated at 37° C. for 2 h. Plates were washed 6times in PBS-TWEEN (polyoxyethylene (20) sorbitan monolaurate(C₅₈H₁₁₄O₂₆)) buffer, after which 100 μl goat anti-bovine IgG-peroxidaseconjugate (SouthernBiotechnology Associates, Inc., Birmingham, Ala.,USA), diluted 1:4000 in PBS-TWEEN (polyoxyethylene (20) sorbitanmonolaurate (C₅₈H₁₁₄O₂₆))-casein, were added to each well. Plates wereincubated for 1 h at 37° C. and washed 6 times. 100 μl peroxidasesubstrate (Kirkegaard and Perry Lab. Inc., Gaithersburg, Md., USA) wereadded to each well and left at room temperature until colour developed.The reaction was stopped by the addition of 2 M sulphuric acid and theplates were read in a Diagnostics Pasteur LP400 plate reader (Sanofi,Mames-1a-Coquette, France) at 450 mm. Results were expressed as the meannet O.D. (after subtraction of the blank reaction) of duplicate wellsassayed on at least two separate occasions.

Example 8 Alternative Immunisation Protocol

Serum antibodies were produced using an antigen prepared according toExample 1. Cow immunisation was as described in example 2 except thatthree injections were given at 2-weekly intervals. Two weeks after thelast of these injections, a blood sample was taken for antibody titredetermination.

Example 9 Production of Serum Antibodies using a Whole Cell Antigen

For the purposes of comparing the immunological response of separatedcell wall antigens and whole cell antigens, serum antibodies were raisedagainst whole cell antigens using the following procedure.

The whole cell antigen was made as follows: 100 ml of Luria broth wasinoculated with E. coli H10407 and incubated at 37° C. overnight withshaking. The resultant culture was centrifuged to pellet the bacteria,which was resuspended in 100 ml of sterile PBS and heated at 100° C. for2.5 hours. The protein concentration was measured using the Bio-Radprotein assay, and adjusted to 1 milligram/ml. Adjuvanting was asdescribed in example 1. Cows were vaccinated using the method of Example7 and blood samples were withdrawn as described in Example 7.

Example 10 Comparison of Whole Cell vs Separated Cell Wall Antigens

Sera derived from group A (18 cows) as per example 7 were run in theElisa assay described in example 7. Sera derived from group B (14 cows)as per example 9 were also run in the Elisa assay of example 6. Theresults were:

Group A Group B EIA units (av) 860 96 EIA units (range) 300-1600 30-300

The titre of antibodies raised using the vaccine of this invention(group A) was significantly greater than the titre of antibodies raisedusing the whole-cell vaccine (group B). This result is surprisingbecause the titre was determined using whole cells immobilised on theElisa plate.

Example 11 Animal Safety

An investigation of 1200 dairy cows vaccinated according to the protocolof example 2 showed no significant welfare problems or site reactions orloss in production.

Example 12

The following assay is based on the use of antibodies raised in thecolostrum of cows to carry out an immunoblot analysis of colonisationfactor. The antibodies are made according to the method of thisinvention, with the proviso that CFA (pilus) antigens are incorporatedin the vaccine.

Buffers & Reagents

10× Tris/Glycine/SDS electrophoresis buffer:

Tris 15 g Glycine 72 g SDS  5 g

Make up to 1 litre with dH₂O. Dilute 1/10 to make “working solution”.

Transfer buffer (100 ml):

Tris 0.56 g Glycine 0.30 g dH₂O 80 ml Methanol 20 ml 10% SDS 360 μl

Combine reagents in the above order and let cool before use.

3× Laemmli buffer (8 ml):

dH₂O 0.8 ml 0.5M Tris-HCl pH 6.8 3.0 ml Glycerol 2.4 ml SDS 0.48 g2-β-Mercaptoethanol* 1.2 ml 0.05% (w/v) Bromophenol Blue 0.6 ml *Addjust before use.

Combine 1 volume Laemmli buffer with 2 vols sample.

Crude Colonisation Factor Preparations

Spread enterotoxigenic E. coli (ETEC) strains on duplicate CFA platesand grow overnight at 37° C. (CF production) and room temperature (CFproduction suppressed). Scrape half a plate of cells into 1 ml PBS andpipette up and down to resuspend. Vortex briefly.

Incubate cell suspensions in a 60° C. waterbath for 30 mins to releasecolonisation factors. Pellet cells by centrifugation (14,000 rpm/20mins) and recover supernatants to fresh tubes.

The crude preparation should contain both colonisation factor and LPSwhere expressed by the strains used.

SDS-PAGE

Pour 15% SDS-polyacrylamide gels with 4% stacking gel as follows:

15% Separating 4% Stacking (4 gels) (4 gels) dH₂O 7.2 ml 6.4 ml 1M TrispH 8.8 5 ml NA 0.5M Tris pH 6.8 NA 2.5 ml ACCUGEL (40%) 7.5 ml 1 ml (asolution of 40% (w/v) acrylamide: bisacrylamide (29:1)) 10% SDS 200 μl100 μl 10% APS 100 μl 50 μl TEMED 10 μl 10 μl Total vol 20 ml 10 ml

Combine 2 vols each crude CF preparation and 1 vol 3× Laemmli buffer togive sufficient final volume ˜25 μl per well required. (i.e. if loadingeach sample on duplicate gels combine 40 I CF and 20 I LB). Boil for 5mins, cool on ice a spin briefly to collect condensation.

Load 20 μl of each sample per well and run gels for 30 mins at 100V(through stacking gel) and 1.5 hours at 150V (dye front will run offend).

Transfer proteins onto nitrocellulose using Trans-Blot apparatus (0.08 Amax per mini gel, 30 mins) and block membranes overnight at 4° C. inPBS+0.1% TWEEN20 (polyoxyethylene (20) sorbitan monolaurate(C₅₈H₁₁₄O₂₆)) (PBS-T)+10% skim milk powder (SMP).

Immunoblot

-   -   Rinse blots with PBS-T (5 mins shaking)

Incubate blots with colostrum from cows hyperimmunised with a vaccinecomprising CFA antigens (100 mg/ml in PBS) diluted 1/200 in PBS-T+5% SMPfor 2 hours with gentle shaking.

Rinse blots and wash with PBS-T for 1×15 mins and 2×5 mins.

Incubate blots with goat anti-bovine IgG-HRPO (1/20,000) in PBS-T+5% SMPfor 1 hour with gentle shaking.

Wash as above, drain excess PBS-T and place damp blots on an overheadsheet. Prepare ECL reagent, drop 1 ml on each gel and leave for 1minute. Place another overhead sheet on top of blots and blot excess ECLreagent ensuring the top where the film will be placed is dry.

Expose the blots to X-ray film for the required amount of time(generally 3 mins initially and then work up or down from there).

Finally, it is understood that various other modifications and/oralterations may be made without departing from the spirit of the presentinvention as outlined herein.

REFERENCES

-   Hitchcock, P. J. and Brown, T. M (1983). Morphological heterogeneity    among Salmonella lipopolysaccharide chemotypes in silver-stained    polyacrylamide gels. Journal of Bacteriology, 154:269-277.-   J. Husu et al. (1993) Production of hyperimmune bovine colostrums    against Campylobacter jejuni. J Appl Bacteriol 74:564-569-   H. Hilpert. (1984) Human Milk Banking, ed. A. F. Williams and J. D.    Baum, Vevey/Raven Press, New York.-   Antibodies-A Laboratory Manual, 1988, ed. By E. Harlow and D. Lane,    Cold Spring Harbour Laboratory Press, p. 298-300.-   A. Cravioto et al. (1982) Hemagglutination activity and colonization    factor antigens I and II in enterotoxigenic and nontoxigenic strains    of Escherichia coli isolated from humans. Infect Immun 36: 189-197.-   J. Lyngby et al. (2002) Biologicals March: 30(i):7-13.    Quantification of lipopolysaccharides.-   S. Y Li et al (2004) Analytical Biochemistry March 1: 326 (1): 1-12.    Comparison of high-performance liquid chromatography and    fluorophore-assisted carbohydrate electrophoresis methods for    analysing peptidoglycan composition of Escherichia coli-   Endotoxins in Health and Disease, eds H. Brade, S Opal, S Vogel, D    Morrison; Marcel Dekker Inc 1999 Chapter 12 and other Chapters.

The invention claimed is:
 1. A method for protection of a human subjectagainst enteric disease caused by gram negative bacteria the methodcomprising: providing O antigen derived polyclonal antibodies raised inthe colostrum of ungulate animals by vaccination of the ungulate animalswith O group serotype antigen, wherein said O antigen is prepared bysubjecting the intact cell walls of gram negative bacteria to shear andseparating the O antigen from cell walls of the gram negative bacteria;and orally administering the O antigen derived polyclonal antibodies tothe human subject, wherein whole cells and separated cell wall fragmentshave been removed from the antigen by centrifugal separation.
 2. Themethod according to claim 1, wherein said antigen is separated fromwalls of gram negative bacteria by homogenisation in a rotor-statorconfiguration, wherein the ratio of the peripheral speed of the rotor(in meters per second) to the rotor-stator gap (in mm) is in the range0.2 to
 20. 3. The method according to claim 2, wherein the ratio of theperipheral speed of the rotor (in meters per second) to the rotor-statorgap (in mm) is in the range of 0.4 to
 12. 4. The method according toclaim 2, wherein the ratio of the peripheral speed of the rotor (inmeters per second) to the rotor-stator gap (in mm) is in the range from0.8 to
 6. 5. The method according to claim 1, wherein the gram negativebacteria are selected from the group consisting of Helicobacter, Vibrio,and Enterobacteriaceae.
 6. The method according to claim 1, wherein thebacteria are E. coli selected from enterotoxigenic E. coli (ETEC),Enterohaemorrhagic E. coli (EHEC), and Enteropathogenic E. coli (EPEC).7. The method according to claim 6, wherein the E. coli is ETEC E. coliand the antigens are selected from the group of O antigens consisting ofO6, O8, O15, O25, O27, O63, O78, O114, O115, O128, O148, O153, and O159.8. The method according to claim 7, wherein the O antigen comprises O78.9. The method according to claim 1, wherein the separated O antigen ispurified using Ammonium Sulphate precipitation to separate outextraneous material.
 10. The method according to claim 9, wherein aninitial precipitation is used to remove extraneous proteins and a finalprecipitation is used to provide the antigens in the precipitate. 11.The method according to claim 1, wherein the antigens separated from thecell walls or wall fragments are subjected to inactivation usingformaldehyde.
 12. The method according to claim 1, wherein saidvaccination is carried out with an adjuvant selected from one or more ofthe group consisting of esters of octodecenoic acid and anhydromannitolin an oily solution, QuilA, and Aluminium Hydroxide.
 13. The methodaccording to claim 1, wherein the said vaccination is carried out with avaccine comprising a group of at least two O antigens, and individual Oantigens are isolated and grown in separate bacterial culture systems,and after separation of the individual O antigens from the bacterialcell walls said antigens are added together in the vaccine.
 14. Themethod according to claim 13, wherein the antigen further comprisesColonization Factor Antigen (CFA) CFA-1 and/or CFA-2.
 15. The method asin claim 14, wherein one group of said group of at least two O antigenscomprises E. coli O78 antigen and CFA-1 pilus antigen, and the othergroup of said group of at least two O antigens comprises antigens chosenfrom the group consisting of O6, O8, O15, O25, O27, O63, O78, O 114,O115, O128, O148, O153, O1598 serotype O antigens and CFA-2 pilusantigen.
 16. The method according to claim 14, wherein the quantity ofCFA antigens per ml in said antigen is greater than the quantity of CFAantigens per ml in a culture of 10⁶ Gram negative bacteria grown underculture conditions conducive to CFA production.
 17. The method accordingto claim 14, wherein the quantity of CFA antigens per ml in said antigenis greater than the quantity of CFA antigens per ml in a culture of 10⁸Gram negative bacteria grown under culture conditions conducive to CFAproduction.
 18. The method according to claim 14, wherein at least 20%of the number of proteins of CFA antigen is dissociated from bacterialcell walls or cell wall fragments.
 19. The method according to claim 14,wherein at least 40% of the number of proteins of CFA antigen isdissociated from the bacterial cell walls or cell wall fragments.